Phosphorylation of 5-bromodeoxycytidine in cells infected with herpes simplex virus

5-Bromodeoxycytidine is phosphorylated to 5-bromodeoxycytidine 5'-monophosphate in extracts of cells infected with herpes simplex virus but not in extracts of uninfected cells. The conversion of 5-bromodeoxycytidine to nucleotides and its utilization for DNA synthesis in uninfected cells occurs by deamination of 5-bromodeoxycytidine to 5-bromodeoxyuridine followed by phosphorylation of 5-bromodeoxyuridine to 5-bromodeoxyuridine 5'-monophosphate. In contrast, in cells infected with herpes simplex virus, 5-bromodeoxycytidine is phosphorylated directly to 5-bromodeoxycytidine 5'-monophosphate, which can then be deaminated to 5-bromodeoxyuridine 5'-monophosphate and incorporated into DNA. These results indicate a difference in the substrate specificity of nucleoside kinases induced by herpes simplex virus and the enzymes present in uninfected cells. It is suggested that this difference in substrate specificity between virus-induced and host-cell enzymes may allow selective chemotherapy of herpes simplex infections with 5-bromo- or 5-iododeoxycytidine.     

Interaction of herpes simplex virus RNase with VP16 and VP22 is required for the accumulation of the protein but not for accumulation of mRNA

The virion host shutoff (vhs) protein encoded by the U(L)41 gene of herpes simplex virus 1 is an endoribonuclease. The enzyme is introduced into the cell during unpackaging of the virion upon entry and selectively degrades mRNA for several hours. The RNase activity ceases after the onset of synthesis of late (gamma) viral proteins. Here we report that vhs protein does not accumulate in cells transiently transfected with only a plasmid encoding the U(L)41 gene. However, vhs does accumulate in cells cotransfected with plasmids expressing two other tegument proteins, VP16 and VP22. vhs does not directly interact with VP22 but, instead, binds VP22 only in the presence of VP16. In contrast to these findings, the amounts of vhs mRNA accumulating in the cells transfected solely with vhs are not significantly different from those detected in cells coexpressing vhs, VP16, and VP22. We conclude from these studies that the steady state of vhs mRNA, reflecting synthesis and turnover of mRNA, is not affected by the interaction of vhs protein with VP16 with VP22. A model is proposed in which the vhs protein may function to sequester mRNAs in compartments inaccessible to the cellular translational machinery and that VP16 and VP22 rescue the mRNAs by interacting with the vhs protein.     

Deoxyribopyrimidine triphosphatase activity specific for cells infected with herpes simplex virus type 1.

Nuclei from baby hamster kidney cells infected with herpes simplex virus type 1 contain a virus-specific deoxyribonucleoside triphosphate degrading activity. The reaction proceeds at 4 degrees C and can thus be distinguished from host enzymes. Under these conditions the enzyme is specific for deoxyribopyrimide triphosphates and catalyzes pyrophosphate cleavage to produce the monophosphates, dUTP being the best substrate followed by dCTP and dTTP. The appearance of the activity after infection parallels that of viral DNA-synthesis-related functions. Of a series of eight temperature-sensitive mutants tested, two (tsD and tsK) exhibit significantly decreased triphosphatase levels after infection at nonpermissive temperature, whereas a viral deoxypyrimidine kinase-deficient mutant induced wild-type levels.     

Genetic engineering of the glucocorticoid receptor by fusion with the herpes viral protein VP22 causes selective loss of transactivation

The development of methods for engineering proteins with novel properties opens the way to manipulating intracellular processes in a therapeutically useful way. Glucocorticoids, acting via glucocorticoid receptors (GR), are potent anti-inflammatory agents, acting to oppose nuclear factor kappa B (NF kappa B) function. The herpes viral protein, VP22, has been reported to confer intercellular trafficking activity on 'cargo' proteins, potentially facilitating gene therapy with intracellular proteins. VP22GR, resulting from the addition of VP22 to the N terminal of GR, was equipotent with the wild-type GR in opposing NF kappa B p65-driven expression of an NF kappa B reporter gene. Surprisingly, VP22GR was incapable of inducing transactivation of positive glucocorticoid reporter genes (MMTV-luc and TAT3-luc). Furthermore, the VP22GR had powerful dominant negative activity on both endogenous and exogenous GR transactivation. VP22GR was cytoplasmic in quiescent cells, and after hormone addition underwent nuclear translocation to share the same distribution as the GR. The ability of the VP22GR to selectively confer and enhance glucocorticoid-dependent transrepression of NF kappa B may be of use therapeutically in e.g. transplant rejection, inflammatory arthritis or asthma.     

Detection of antigen to herpes simplex virus in cerebrospinal fluid from patients with herpes simplex encephalitis

Cerebrospinal fluid (CSF) specimens were obtained from patients with presumed herpes simplex encephalitis who underwent brain biopsy for diagnostic confirmation. Coded CSF specimens were fixed on nitrocellulose filter paper and probed with a pool of monoclonal antibodies directed against four herpes simplex virus glycoproteins (gB, gC, gD, and gE). Herpes simplex virus antigen was detected in 35 of 40 specimens obtained from 26 biopsy-positive patients. In contrast, only three of 25 specimens from 17 biopsy-negative patients gave positive results by this assay. An additional 30 CSF specimens from patients with proven bacterial and fungal infections were all negative by this assay. For all specimens tested, the sensitivity and specificity of the assay were both 88%. However, when results were evaluated by patient, the sensitivity was 92% (24 of 26) with a specificity of 82% (14 of 17). Among specimens collected one week or later after disease onset, the sensitivity was 100%, with a specificity of 93%.     

Pathogenesis of herpes simplex labialis. I. Replication of herpes simplex virus in cultures of epidermal cells from subjects with frequent recurrences

Primary cultures were established with epidermal cells from the skin of 11 patients with frequent episodes of herpes simplex labialis and 13 control subjects with titers of neutralizing antibody to herpes simplex virus (HSV) type 1 but no history of herpetic disease. Confluent monolayers were exposed to HSV type 1 strain E115, and the infection was monitored by assay of the rate of virus appearance in the culture medium. The mean slope of the virus growth curves ([log10 pfu/ml]/log10 hr) was 9.0 in cultures from patients vs. 9.5 in cultures from controls, and the respective mean titers of virus 53 hr after infection were 10(6.8) and 10(6.5) (differences not statistically significant). Genetically controlled host factors may play some role in the clinical response to HSV infection, but variation in the susceptibility of epidermal cells, the natural target for HSV, is not one of the critical determinations.     

ICP0 enables and monitors the function of D cyclins in herpes simplex virus 1 infected cells

The herpes simplex virus 1 ICP0 is a regulatory protein. Early in infection ICP0 localizes in ND10 bodies and performs two functions: As an E3 ligase in conjunction with E2 UbcH5a conjugating enzyme, it degrades the ND10 components PML and SP100. Concurrently, it suppresses the silencing of viral DNA by dispersing the HDAC1/CoREST/REST/LSD1 repressor complex. Subsequently, ICP0 is exported to the cytoplasm. In cells treated with HDAC inhibitors or transfected with irrelevant DNA, the export is delayed in a DNA dose-dependent fashion. Here, we follow up an observation that ICP0 binds cyclin D3 and that ICP0 mutants unable to bind cyclin D3 are not exported. Moreover, in infected cells cdk4 is activated, but cdk2 is not. We report that (i) cyclin D1, D2, or D3 colocalize with ND10 bodies and ICP0 early in infection and ultimately become incorporated into viral replication compartments, (ii) each of the D cyclins partially rescues ΔICP0 mutants, and (iii) inhibition of cdk4 by inhibitor I sequesters ICP0 in the nucleus. A key finding is that overexpression of cyclin D3 enables the transport of ICP0 to the cytoplasm. We conclude that (i) ICP0 facilitates the recruitment of cyclin D3 to the sites of viral DNA synthesis, (ii) until its functions are completed, ICP0 is retained in the nucleus, and (iii) a common signal that results in the export of ICP0 to the cytoplasm is the accumulation of a viral DNA-synthesis-dependent late protein.     

New observations regarding killing of fibroblasts infected with herpes simplex virus: cooperation between elutable factor and peripheral mononuclear cells

With a standard chromium release assay, natural killing (NK) activity of peripheral mononuclear cells (PMCs) from 28 individuals was compared based on the ability of sera to support antibody-dependent cell-mediated cytotoxicity (ADCC) for cells infected with herpes simplex virus (HSV). PMCs from all 20 individuals whose sera produced ADCC were capable of killing HSV-infected cells compared with none of the PMCs from the eight individuals whose sera did not produce ADCC (mean specific release, 33.2% vs. 6.8%). Results could not be explained by contaminating serum in the assay or by ineffective NK by the PMCs from the eight negative subjects because many of them killed the k562 myeloid cell line as effectively as PMCs from other individuals. In addition, NK could be eliminated by preincubation of effector cells at 37 C, and the capacity to kill by PMCs could be reconstituted by incubation in serum. Killing was more a function of source of serum rather than source of cells.     

The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors.

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.     

Interaction of herpes simplex virus 1 origin-binding protein with DNA polymerase alpha.

The herpes simplex virus 1 (HSV-1) genome encodes seven polypeptides that are required for its replication. These include a heterodimeric DNA polymerase, a single-strand-DNA-binding protein, a heterotrimeric helicase/primase, and a protein (UL9 protein) that binds specifically to an HSV-1 origin of replication (oris). We demonstrate here that UL9 protein interacts specifically with the 180-kDa catalytic subunit of the cellular DNA polymerase alpha-primase. This interaction can be detected by immunoprecipitation with antibodies directed against either of these proteins, by gel mobility shift of an oris-UL9 protein complex, and by stimulation of DNA polymerase activity by the UL9 protein. These findings suggest that enzymes required for cellular DNA replication also participate in HSV-1 DNA replication.     

Uptake of [125I]iododeoxycytidine by cells infected with herpes simplex virus: a rapid screening test for resistance to acyclovir

A rapid screening test for resistance to acyclovir, mediated by a lack of thymidine kinase (TK) activity in herpes simplex virus (HSV), was developed by utilizing the uptake of [125I]iododeoxycytidine (IdC) by infected Vero cells. Cells infected with TK+ virus demonstrate uptake of IdC within 3 hr of infection. The assay can detect as few as 690 pfu of virus. Cells infected with TK virus have an uptake of IdC similar to that of uninfected cells, whereas cells infected with TK+ generally have an uptake greater than 10 times that of uninfected cells if tested when obvious viral cytopathic effect is present. All 19 clinical HSV isolates tested were correctly identified as TK+. Of 17 blinded HSV isolates tested, all six TK- isolates were correctly identified. A single strain with an ID50 of 3.4 micrograms/ml and an altered TK substrate specificity was incorrectly classified as TK+. The assay is a useful, rapid screening test for viral TK activity.     

Predominance of herpes simplex virus type 1 from patients with genital herpes in Nova Scotia

The epidemiology of genital herpes is changing with evidence to suggest an increasing incidence of herpes simplex virus type 1 (HSV-1) infections. The results of 6529 HSV genital cultures taken between April 1998 and December 2001 were reviewed. overall, HSV-1 was recovered more often than HSV-2; 1213 versus 1045. This trend was particularly striking in young women 30 years of age or less, in whom 70.8% of isolates were HSV-1. In men of the same age range, 45% of isolates were HSV-1. The proportion of women with HSV-1 declined from 73.7% in those younger than 31 years of age to 4.5% in those older than 60 years of age.These observations have important implications. The decline in the relative proportion of HSV-1 isolates from young adults may be the result of changing sexual practices, changing susceptibility or increased exposure to HSV-1 during vaginal intercourse. In this setting HSV-2 vaccines may be less likely to produce the desired reduction in the overall prevalence of genital herpes infections.Key Words: Genital herpes, Herpes simplex virus type-1Many factors have contributed to the changing epidemiology of sexually transmitted diseases (STDs) in the past decade. Changes in sexual practices, improved diagnosis and more effective treatments have dramatically reduced the incidence of chlamydia, gonorrhea and syphilis (1). On the other hand, in many countries the incidence of genital herpes (GH) infections has not declined. In the United States, the number of physician office visits for GH has increased markedly since 1966 (1). There are likely several reasons why significant declines have not been observed: the period of infectivity may extend over many years, GH may be less effectively prevented by condom use, diagnosis of subclinical infections is technically difficult, and no curative treatments are available. Many patients have numerous asymptomatic recurrences and are unable to identify periods of increased infectivity.There is evidence that the relative prevalence of herpes simplex virus type 1 (HSV-1) and HSV-2 genital infections may be changing. Recent studies from Scandinavia and the United States suggest that HSV-1 infections are increasingly prevalent (2-5). We reviewed the results of genital cultures performed at our hospital over a 30-month period to determine whether this is also the case in Nova Scotia.     

Cells infected with herpes simplex virus 1 export to uninfected cells exosomes containing STING,viral mRNAs,and microRNAs

STING (stimulator of IFN genes) activates the IFN-dependent innate immune response to infection on sensing the presence of DNA in cytosol. The quantity of STING accumulating in cultured cells varies; it is relatively high in some cell lines [e.g., HEp-2, human embryonic lung fibroblasts (HEL), and HeLa] and low in others (e.g., Vero cells). In a preceding publication we reported that STING was stable in four cell lines infected with herpes simplex virus 1 and that it was actively stabilized in at least two cell lines derived from human cancers. In this report we show that STING is exported from HEp-2 cells to Vero cells along with virions, viral mRNAs, microRNAs, and the exosome marker protein CD9. The virions and exosomes copurified. The quantity of STING and CD9 exported from one cell line to another was inoculum-size–dependent and reflected the levels of STING and CD9 accumulating in the cells in which the virus inoculum was made. The export of STING, an innate immune sensor, and of viral mRNAs whose major role may be in silencing viral genes in latently infected neurons, suggests that the virus has evolved mechanisms that curtail rather than foster the spread of infection under certain conditions.The stimulator of IFN genes (STING) is a sensor of cytoplasmic DNA and activates immune responses to intracellular pathogens (1–3). Knockout of STING exacerbates the pathogenicity of herpes simplex virus (HSV-1) and of other pathogens in mice (2, 3). Two observations reported earlier add to the role of STING in HSV-1 infected cells. Specifically, (i) STING was readily detectable and stable in four different cell lines infected with wild-type virus (4). The stability of STING in cells infected with an HSV-1 mutant lacking the gene encoding ICP0 (infected cell protein no. 0), a key viral regulatory protein, varied. STING was stable in ΔICP0 mutant virus infected cells derived from normal tissues but was rapidly degraded in infected cells derived from human cancers (4). Implicit in this observation is that ICP0 is required to stabilize STING, although STING could also be stabilized in the absence of ICP0 by a cellular function. (ii) Depletion of STING increased wild-type virus yield 10-fold in cells derived from normal tissues but decreased the yield by the same amount in cells derived from human cancer (4). Thus, at least in cells derived from normal tissues, HSV-1 expresses ICP0 that enables the stabilization of STING even though STING is inimical to virus growth. The results of these studies suggest that HSV-1 recruits STING for a specific function, even though the persistence of STING is not beneficial to virus growth, at least in cells derived from normal tissues (4).Here we report that STING, along with viral RNAs contained in structures that coprecipitate with exosome marker proteins, is exported from the cells in which virus is produced to uninfected cells. The quantities introduced into uninfected cells are dose-dependent and reflect the amounts produced in donor cells.Cells continuously secrete a large number of microvesicles, macromolecular complexes, and small molecules into the extracellular space (5, 6). Of the secreted microvesicles, exosomes are 30–120 nm in diameter, containing nucleic acid and proteins, and are perceived to be carriers of this cargo between diverse locations. They are distinguished in their genesis by being budded into endosomes to form multivesicular bodies (MVBs) in the cytoplasm. The exosomes are released to extracellular fluids by fusion of these multivesicular bodies with the cell surface, resulting in secretion (7–10).Several pathogens use the exosomes to manipulate their microenvironment (11, 12). Viruses, especially small retroviruses such as HIV, use the exosome pathway for egress and immune evasion (13–15). The hepatitis C virus uses exosomes for invasion and spread (16–18). EBV-infected cells release exosomes containing the latent membrane protein 1 to induce T-cell anergy (19–23). In the case of human cytomegalovirus the exosomes carrying viral antigens exacerbate the transplant rejection process (24). It is likely that viruses that establish long-term, latent, or chronic infections modulate exosomes to enhance their persistence (11).Here we report that the exosomes secreted by HSV-1–infected cells deliver to uninfected cells the innate immune sensor STING along with viral RNAs. We speculate that in the long run the strategy serves the virus to fulfill its mission: to spread effectively from person to person.     

Native 3D intermediates of membrane fusion in herpes simplex virus 1 entry

The concerted action of four viral glycoproteins and at least one cellular receptor is required to catalyze herpes simplex virus 1 entry into host cells either by fusion at the plasma membrane or intracellularly after internalization by endocytosis. Here, we applied cryo electron tomography to capture 3D intermediates from Herpes simplex virus 1 fusion at the plasma membrane in their native environment by using two model systems: adherent cells and synaptosomes. The fusion process was delineated as a series of structurally different steps. The incoming capsid separated from the tegument and was closely surrounded by the cortical cytoskeleton. After entry, the viral membrane curvature changed concomitantly with a reorganization of the envelope glycoprotein spikes. Individual glycoprotein complexes in transitional conformations during pore formation and dilation revealed the complex viral fusion mechanism in action. Snapshots of the fusion intermediates provide unprecedented details concerning the overall structural changes occurring during herpesvirus entry. Moreover, our data suggest that there are two functional “poles” of the asymmetric herpesvirion: one related to cell entry, and the other formed during virus assembly.     

Autophagic flux without a block differentiates varicella-zoster virus infection from herpes simplex virus infection

Autophagy is a process by which misfolded and damaged proteins are sequestered into autophagosomes, before degradation in and recycling from lysosomes. We have extensively studied the role of autophagy in varicella-zoster virus (VZV) infection, and have observed that vesicular cells are filled with >100 autophagosomes that are easily detectable after immunolabeling for the LC3 protein. To confirm our hypothesis that increased autophagosome formation was not secondary to a block, we examined all conditions of VZV infection as well as carrying out two assessments of autophagic flux. We first investigated autophagy in human skin xenografts in the severe combined immunodeficiency (SCID) mouse model of VZV pathogenesis, and observed that autophagosomes were abundant in infected human skin tissues. We next investigated autophagy following infection with sonically prepared cell-free virus in cultured cells. Under these conditions, autophagy was detected in a majority of infected cells, but was much less than that seen after an infected-cell inoculum. In other words, inoculation with lower-titered cell-free virus did not reflect the level of stress to the VZV-infected cell that was seen after inoculation of human skin in the SCID mouse model or monolayers with higher-titered infected cells. Finally, we investigated VZV-induced autophagic flux by two different methods (radiolabeling proteins and a dual-colored LC3 plasmid); both showed no evidence of a block in autophagy. Overall, therefore, autophagy within a VZV-infected cell was remarkably different from autophagy within an HSV-infected cell, whose genome contains two modifiers of autophagy, ICP34.5 and US11, not present in VZV.VZV induces macroautophagy (hereafter referred to as autophagy) in skin cells within the typical exanthem associated with either primary VZV infection (varicella or chickenpox) or VZV reactivation (herpes zoster or shingles). During prior studies, the extent of autophagy was gauged by enumeration of autophagosomes by both 2D and 3D microscopy (1, 2). The usual number of autophagosomes seen by 3D animation was 100 per infected cell, but sometimes approached 200 per cell. In contrast, a typical nonstressed cell usually has fewer than 4 autophagosomes (3, 4). When monolayers were inoculated with VZV-infected cells, the traditional method for VZV infection, autophagy was again easily seen after enumeration of autophagosomes and immunoblotting for the LC3-phosphatidylethanolamine conjugate (LC3-II). Again these results suggested that autophagic flux was present during VZV infection in cultured cells.As part of a more extensive assessment of autophagy after VZV-induced cellular stress, we have now investigated autophagy in infected human skin xenografts from the SCID mouse model of VZV infection. This model is the most accurate representation of the skin manifestation of varicella in the human host (5, 6). Finally, we addressed an important point about the nature of VZV-induced autophagy. Because the number of autophagosomes seen in the human vesicle cells from varicella and herpes zoster patients is so high, the question has arisen whether there is a late block in the maturation of autophagosomes to autolysosomes. In this report, we demonstrate that (i) autophagy induced by VZV infection is related to the overall stress to the cell, namely, a higher inoculum leads to greater autophagy; and that (ii) autophagosomes induced during VZV infection mature into autolysosomes without an obvious block before final maturation. The autophagic flux assay results confirm that VZV infection induces autophagy that proceeds to completion, possibly allowing the cell to alleviate the cellular stress caused by the viral infection (7). In previous work, we showed that inhibition of autophagy led to a significant decrease in VZV titer. Overall, therefore, autophagy within a VZV-infected cell is remarkably different from autophagy within an HSV-infected cell, an alphaherpesvirus whose genome contains two modifiers of autophagy, ICP34.5 and US11 (8–13). In contrast, autophagy appears to be proviral in the life cycle of VZV.